Trichomonas vaginalis, a parasite, causes one of the most common non-viral sexually transmitted disease in humans, trichomoniasis. Trichomoniasis is a serious disease that causes vaginitis in women and urethritis in men. It has been linked to infertility, preterm delivery, low birth weight infants and cervical cancer. T. vaginalis may be emerging as one of the most important cofactors in amplifying HIV transmission, especially in African-American communities of the United States. The long-term goal of this proposal is to elucidate the mechanisms of T. vaginalis pathobiology at the molecular and cellular levels. We have developed an in vitro culture system of human vaginal epithelial cells (HVECs) to study parasite-host cell interaction. T. vaginalis infection of host cells in vitro induces cell destruction. The soluble fraction (SF) elaborated (secreted/released) by the parasites induces apoptosis in the HVECs in a species-specific manner. The partially purified active agent(s) from T. vaginalis-SF contains a cysteine protease(s) that appears to be responsible for inducing apoptosis in HVECs. The specific aims are: 1) to purify and characterize the protease(s) that induce apoptosis; including, cloning, sequencing, expression of recombinant protein and express mutants of the active molecule(s); 2) study the physiology/cell biology of the parasite molecules with respect to biosynthesis, secretion, and activation of the apoptotic protease(s) by T. vaginalis; and 3) identify the initial events leading to HVEC apoptosis triggered by the parasite molecule (s), including cell surface death receptors. The active toxic agents will be purified by a series of chromatographic procedures. Each step will be monitored using both cytotoxicity and protease assays. The purified material will be subjected to proteolytic digestion and isolated peptides will be sequenced. Sequence information will be used to clone the cDNA. The cDNA will then be used to produce recombinant protein, which will be tested in functional assays to demonstrate that the cloned protein induces HVEC apoptosis. Antibodies (Abs) will be generated against active protease(s) to study the optimal release of cytotoxic agent under a variety of conditions and to show if it is synthesized in a soluble form or associated with membranes. Antagonist Abs against death receptors will be utilized with the purified agent to elucidate which of the receptors is involved in triggering apoptotic pathways. Activation of caspases-8, -9, and -3 will be monitored by Western blot/flow cytometry analyses using specific Abs and quantified using specific peptide substrates. Knowledge of the molecular understanding of host cell pathology induced by parasite will reveal new strategies/effective treatment for trichomoniasis.